Structure-based redesign of an edema toxin inhibitor

Abstract

Edema factor (EF) toxin of Bacillus anthracis (NIAID category A), and several other toxins from NIAID category B Biodefense target bacteria are adenylyl cyclases or adenylyl cyclase agonists that catalyze the conversion of ATP to 3',5'-cyclic adenosine monophosphate (cAMP). We previously identified compound 1 (3-[(9-oxo-9H-fluorene-1-carbonyl)-amino]-benzoic acid), that inhibits EF activity in cultured mammalian cells, and reduces diarrhea caused by enterotoxigenic Escherichia coli (ETEC) at an oral dosage of 15μg/mouse. Here, molecular docking was used to predict improvements in potency and solubility of new derivatives of compound 1 in inhibiting edema toxin (ET)-catalyzed stimulation of cyclic AMP production in murine monocyte-macrophage cells (RAW 264.7). Structure-activity relationship (SAR) analysis of the bioassay results for 22 compounds indicated positions important for activity. Several derivatives demonstrated superior pharmacological properties compared to our initial lead compound, and are promising candidates to treat anthrax infections and diarrheal diseases induced by toxin-producing bacteria.

Figure 1

Structures for the lead compound 1 and derivatives 2~22 analyzed in this work.

Figure 2

A: Overlay of the docked conformation of 1 with the position of 3d’-ATP from the crystal structure (in 1K90.pdb). B: dotted spheres: pharmacophores used to screen ZINC database.

Figure 3

Effect of protonation state of the benzene side chain sidechain –C(=NH)NH₂ on potential interactions with EF residues. Docking poses are shown for 22 when neutral (A) or positively charged (B). In A, this group interacts with the metal ion, the backbone oxygen atom of Lys346, Gly347, Val50 and the carboxyl group of Asp491, while in B it interacts with the backbone oxygen atoms of Lys346, Gly347, Val50 and the carboxyl group of Asp491.

Figure 4

Schematic drawing, showing the position of 3’dATP (thick ball and stick figure) in the 1K90 crystal structure, overlaid with the docking pose of 22 (green thin ball and stick figure). The silver point indicates the position of the metal ion, the blue and red points indicate positive and negative charged groups, respectively, from EF sidechains or backbone oxygens. The blue circle indicates a positively charged region, where negatively charged groups, for example, the triphosphate of ATP, could be added to enhance inhibitory activity. The red circle indicates a negatively charged region formed by the backbone oxygen atoms of Lys346, Gly347 Val350 and the carboxyl group of Asp491, where the –C(=NH)NH₂ sidechain of the 3-carbamimidoylphenyl group of 22 lies. The Green circle indicates a hydrophobic region formed by the side chains of Leu348, and other residues that are not explicitly shown, where the purine of ATP and the fluorenone of 22 lie.